Project Summary/Abstract - Epigenetic HIV Silencing in Macrophages Antiretroviral therapy (ART) is highly effective in HIV infection but has substantial limitations, and strategies to achieve an aviremic state without ART (?functional cure?) are a focus of great interest. A potential approach to prevent persistent viral reservoirs from giving rise to replicating virus once ART is stopped is to silence HIV in these reservoirs so infection cannot ?re-ignite?. Infected macrophage lineage cells serve as the main long-term reservoir for HIV in the CNS, which is believed to be a ?sanctuary site? where virus can persist even if extra- CNS reservoirs are purged. Furthermore, long-lived infected macrophages may continue to produce low levels of virus, which contributes to HIV-associated neurocognitive disorders (HAND). Thus, silencing HIV in macrophages may both contribute to ?functional cure?, and ameliorate HAND in the setting of ART suppression. HIV integrates into the host cell genome and is subject to positive and negative epigenetic regulation. Much work has been done on epigenetic control of HIV in T cells, but less is known in macrophages. Chromatin organization and transcriptional regulation is highly cell and context-specific. Our scientific premise is that macrophages in the CNS fail to epigenetically suppress HIV and serve as a long-term reservoir. We hypothesize that macrophage-specific enhancer-promoter interactions regulate persistent HIV transcription activity, and that small molecules can reprogram epigenetic regulation of HIV infected macrophages to establish long-term silencing of the integrated HIV genome. To this end, we have established a primary macrophage model for high-throughput screening of small molecule modulators of epigenetic enzymes, and provide preliminary evidence for involvement of dioxygenase enzymes involved in histone demethylation. In Phase 1, we will optimize primary & secondary assays for a high-throughput screening campaign to identify small molecule epigenetic regulators of HIV-1 expression in primary human macrophages; test the role of candidate epigenetic modulators, especially histone & DNA demethylases that may prevent HIV silencing; and further investigate molecular mechanisms that regulate persistent HIV transcription in macrophages. In Phase 2, we will perform a high-throughput screen to develop small molecules to silence HIV-1 in macrophages using industry-standard milestone-driven drug discovery pipeline to identify bioactive chemotypes; validate lead compounds with primary HIV isolates & myeloid cell types; advance drug-like properties of candidate hits through medicinal chemistry; and define molecular targets and conduct mechanism of action studies. At the end of Phase I we will have a robust high-throughput primary cell-based screen, orthogonal validation assays to confirm on-target hits, and further insight into macrophage-specific epigenetic factors regulating HIV. At the end of Phase 2, we will complete a high-throughput screen of several structurally diverse small molecule libraries, identify & validate hits, and select and advance lead candidates. We anticipate this work will lead to identification of novel small molecules as a basis for HIV silencing approaches focused on the CNS reservoir.